Abstract

Globally, marine sediments are a vast repository of organic matter which is degraded through various microbial pathways, including polymer hydrolysis and monomer fermentation. The sources, abundances, and quality (i.e. labile or recalcitrant) of the organic matter and the composition of the microbial assemblages vary between sediments. Here, we examine new and previously published sediment metagenomes from the Baltic Sea and the nearby Kattegat to determine connections between geochemistry and the community potential to degrade organic carbon. Diverse organic matter hydrolysis encoding genes were present in sediments between 0.25 to 67 meters below seafloor, and were in higher relative abundances in those sediments that contained more organic matter. New analysis of previously published metatranscriptomes demonstrated that many of these genes were transcribed in two organic-rich Holocene sediments. Some of the variation in deduced pathways in the metagenomes correlated to carbon content and depositional conditions. Fermentation-related genes were found in all samples, and encoded for multiple fermentation strategies. Notably, genes conferring alcohol metabolism were amongst the most abundant of these genes, indicating this is an important but underappreciated aspect of sediment carbon cycling. This study is a step towards a more complete understanding of microbial food webs and the impacts of depositional facies on present sedimentary microbial communities.

Abstract

To describe a microbe’s physiology, including its metabolism, environmental roles, and growth characteristics, it must be grown in a laboratory culture. Unfortunately, many phylogenetically novel groups have never been cultured, so their physiologies have only been inferred from genomics and environmental characteristics. Although the diversity, or number of different taxonomic groups, of uncultured clades has been studied well, their global abundances, or numbers of cells in any given environment, have not been assessed. We quantified the degree of similarity of 16S rRNA gene sequences from diverse environments in publicly available metagenome and metatranscriptome databases, which we show have far less of the culture bias present in primer-amplified 16S rRNA gene surveys, to those of their nearest cultured relatives. Whether normalized to scaffold read depths or not, the highest abundances of metagenomic 16S rRNA gene sequences belong to phylogenetically novel uncultured groups in seawater, freshwater, terrestrial subsurface, soil, hypersaline environments, marine sediment, hot springs, hydrothermal vents, nonhuman hosts, snow, and bioreactors (22% to 87% uncultured genera to classes and 0% to 64% uncultured phyla). The exceptions were human and human-associated environments, which were dominated by cultured genera (45% to 97%). We estimate that uncultured genera and phyla could comprise 7.3 × 1029 (81%) and 2.2 × 1029(25%) of microbial cells, respectively. Uncultured phyla were overrepresented in metatranscriptomes relative to metagenomes (46% to 84% of sequences in a given environment), suggesting that they are viable. Therefore, uncultured microbes, often from deeply phylogenetically divergent groups, dominate nonhuman environments on Earth, and their undiscovered physiologies may matter for Earth systems.

Abstract

Difficulty quantifying biogeochemically significant microbes in marine sediments limits our ability to assess interspecific interactions, population turnover times, and niches of uncultured taxa. We incubated surface sediments from Cape Lookout Bight, North Carolina USA, anoxically at 21°C for 122 days. Sulfate decreased until day 68, after which methane increased, with hydrogen concentration consistent with predicted values of an electron donor exerting thermodynamic control. We measured turnover times using two relative quantification methods, quantitative PCR (qPCR) and the product of 16S gene read abundance and total cell abundance (FRAxC, for fraction of read abundance times cells), to estimate population turnover rates of uncultured clades. Most 16S rRNA reads were from deeply-branching uncultured groups and ∼ 98% of 16S rRNA genes did not abruptly shift in relative abundance when sulfate reduction gave way to methanogenesis. Uncultured Methanomicrobiales and Methanosarcinales increased at the onset of methanogenesis with population turnover times estimated from quantitative PCR (qPCR) at 9.7 ± 3.9 and 12.6 ± 4.1 days, respectively. These were consistent with FRAxC turnover times of 9.4 ± 5.8 and 9.2 ± 3.5 days, respectively. Uncultured Syntrophaceae, which are possibly fermentative syntrophs of methanogens, and uncultured Kazan-3A-21 archaea also increased at the onset of methanogenesis with FRAxC turnover times of 14.7 ± 6.9 and 10.6 ± 3.6 days. Kazan-3A-21 may therefore either perform methanogenesis or form a fermentative syntrophy with methanogens. Three genera of sulfate reducing bacteria, Desulfovibrio sp., Desulfobacter sp., and Desulfobacterium sp. increased in the first 19 days before declining rapidly during sulfate reduction. We conclude that population turnover times on the order of days can be measured robustly in organic-rich marine sediment, and the transition from sulfate-reducing to methanogenic conditions only stimulates growth in a few clades directly involved in methanogenesis, rather than the whole microbial community.

Abstract

Microbial life in the deep subsurface biosphere is taxonomically and metabolically diverse, but it is vigorously debated whether the resident organisms are thriving (metabolizing, maintaining cellular integrity, and expressing division genes) or just surviving. As part of Integrated Ocean Drilling Program (IODP) Expedition 347: Baltic Sea Paleoenvironment, we extracted and sequenced RNA from organic carbon-rich, nutrient-replete, and permanently anoxic sediment. In stark contrast to the oligotrophic subsurface biosphere, Baltic Sea Basin samples provided a unique opportunity to understand the balance between metabolism and other cellular processes. Targeted sequencing of 16S rRNA transcripts showed Atribacteria (an uncultured phylum) and Chloroflexi to be among the dominant and the active members of the community. Metatranscriptomic analysis identified methane cycling, sulfur cycling, and halogenated compound utilization as active in situ respiratory metabolisms. Genes for cellular maintenance, cellular division, motility, and antimicrobial production were also transcribed. This indicates that microbial life in deep subsurface Baltic Sea Basin sediments was not only alive, but thriving.

Acquisition Description

Samples were collected with SCUBA divers at Cape Lookout Bight, North Carolina (34.6205°N, 76.5500°W), October 2, 2013. Thirty 20 cm PVC push cores were collected, capped, refrigerated, and then returned to the lab on ice in Tennessee within 48 hrs. Bubbles of methane were released from the sediments as each core was taken, indicating the presence of methane at the surface of the core. Using a plunger inserted from the bottom the first three centimeters of sediment taken from each core was placed in a 2L Erlenmeyer flask by way of a funnel. About ten core tubes were needed to fill each of the three flasks to 1.5 liters of sediment. About 100 ml of slurried sediment were autoclaved and incubated alongside the experiments under anoxic conditions as a negative control. Cell counts, hydrogen, sulfate, and methane were measured for the negative control on day 260. All measurements described below were performed for 18 weekly timepoints for all three incubations (0-122 days). An additional timepoint at 802 days was taken for 16S rRNA gene sequence analysis only.

Geochemistry

Each of the three flasks was fitted with a custom butyl rubber stopper with a hole drilled through the center to accommodate a wide bore (6 mm) glass and Teflon stopcock for the removal of samples. Two 18-gauge needles with stainless steel stopcocks were inserted into the stopper as well. Using the luer-lock fitting on the needles, ultra high purity nitrogen gas (99.999%) that had been scrubbed of oxygen using heated copper fillings was flowed through the bottles using the second needle for the outflow to make the headspace anoxic. Incubation 3 was then flushed with ≥ 99.0% methane gas (Sigma-Aldrich, St. Louis, MO). Then all the ports were closed and the flasks inverted so that sediment covered the gas ports, stopper, and stopcock, and placed in a ring stand at constant room temperature (21.4°C) in the dark.

The incubations were turned over once every seven days just before sampling. Prior to gas sampling, 2 ml of anoxic N2 gas (99.999%) was used to blow the needle clear of sediment. Hydrogen and methane gas samples were collected in glass gastight Hamilton syringes using the steel needle ports in the custom stopper. About 32 ml of sediment was removed through the glass and Teflon stopcock using a sterile 60 ml plastic catheter tip syringe. From this, two 15ml conical centrifuge tubes were filled and capped, one used for porewater analysis and the other frozen at -80°C for later molecular analysis. One ml of sediment was placed in a 2 ml screw cap tube to be fixed and used for cell counts as described below. After sampling, 30ml of oxygen- and hydrogen-scrubbed N2 was injected into the bottle to replace the lost volume. The 15 ml tube destined for porewater analysis was centrifuged at 5000 xG for 5 minutes. A syringe was used to remove the supernatant not in contact with the air. The porewater was then filtered using a 0.2 μm syringe filter into 100 μl of 10% HCl to a final volume of 1 ml. Porewater sulfate content was determined by ion chromatography (Dionex, Sunnyvale, CA), with the remaining porewater used for determining the pH.

500µl of headspace gas was injected into a Peak Performer 1 Reducing Compound Photometer (Peak Laboratories, Mountain View, CA). Premixed hydrogen ppm lab bottles (Sigma-Aldrich, St. Louis, MO) were used as standards. Hydrogen was assumed to be equilibrated between headspace and porewater, since the equilibration time for Cape Lookout Bight sediments is < 2 days (Hoehler et al. 1998), which is less than the 7 days between timepoints in our study. Therefore, gas phase partial pressures were converted to aqueous hydrogen concentrations using the solubility coefficient of hydrogen corrected for salinity of 35 ppt and temperature of 21.4°C (Crozier & Yamamoto 1980). Methane was determined by using injected 500 µl of gas from the headspace into an evacuated glass bottle to be later analyzed on a gas chromatograph with a flame ionized detector (Agilent, Santa Clara, CA). Methane concentrations were not assumed to be equilibrated with the aqueous phase, therefore concentrations are presented as partial pressures.

Cell quantification

Total cell counts were determined by direct epifluorescence microscopy SYBRGold DNA stain (Invitrogen, Carlsbad, CA). Sediments were sonicated at 20% power for 40 seconds to disaggregates cells from sediments and diluted 40-fold into PBS prior to filtration onto a 0.2 μm polycarbonate filter (Fisher Scientific, Waltham, MA) and mounted onto a slide. An autoclaved sediment sample was used as a negative control.

Quantitative PCR

DNA was extracted from sediment samples frozen at -80°C using the Fast DNA kit for Soil (MP Bio, Santa Ana, CA). Negative controls of sterilized sediment and a blank water extract were used as well. Quantitative PCR was used to determine 16S rRNA gene copy numbers of several taxa with Quantifast SYBRGreen kit (Qiagen) on a BioRad IQ5 machine. DNA standards were prepared from either existing stocks (Lloyd et al., 2011) or from TOPO plasmids (Invitrogen, Carlsbad, CA) containing PCR amplified 16S rRNA gene products of closely related relatives in the clades Methanomicrobiales and Methanosarcinales synthesized by Invitrogen (accession # AB236118 and AB679168 respectively). Standards were quantified using Hoechst dye in a flourimeter (Hoefer, Holliston, MA). 16s rRNA specific primers Methanomicrobiales and Methanosarcinales primers were selected to have good coverage of the taxa (Narihiro and Sekiguchi, 2011).

16S Ribosomal RNA Gene Amplicons

Extracted DNA was used for 16S rRNA gene amplicon analysis. The V4 region of each DNA extraction was amplified using primers 806r and 515f (Caporaso et al., 2012), as a universal primer pair for Bacteria and Archaea. Library preparations via Nexterra kit and sequencing using an Illumina MiSeq were performed at the Center for Environmental Biotechnology at the University of Tennessee in Knoxville. The Mothur MiSeq Standard Operating Procedure was used to make contigs of bidirectional sequences, cluster operational taxonomic units (OTUs) at 97% similarity, and classify them with the Silva reference set 119 (Schloss et al., 2009, Pruesse et al., 2007). 26.4% of unique sequences were removed as chimeric and then approximately 5% of total sequences were removed for failing to classify at the domain level. Reads were normalized against the sum of reads classified as bacteria and archaea.

Analyses of time course patterns of various microbial taxa were considered only for those with more than 20 reads when summed from the 18 timepoints from each of the three incubations, leaving between 593 and 669 genus-level clades of bacteria and archaea across the three incubations. Total reads ranged from 20,922 to 329,380 for the 54 libraries. 16S rRNA sequences were normalized to total classifiable bacterial and archaeal reads. ]

Processing Description

Raw data has been converted to environmentally relevant units as calculated from appropriate standards. Data that is below detection limits is flagged as no data (nd).

Instruments

Ion chromatography is a form of liquid chromatography that measures concentrations of ionic species by separating them based on their interaction with a resin. Ionic species separate differently depending on species type and size. Ion chromatographs are able to measure concentrations of major anions, such as fluoride, chloride, nitrate, nitrite, and sulfate, as well as major cations such as lithium, sodium, ammonium, potassium, calcium, and magnesium in the parts-per-billion (ppb) range. (from http://serc.carleton.edu/microbelife/research_methods/biogeochemical/ic.html)

Instrument separating gases, volatile substances, or substances dissolved in a volatile solvent by transporting an inert gas through a column packed with a sorbent to a detector for assay. (from SeaDataNet, BODC)

Instruments that generate enlarged images of samples using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption of visible light. Includes conventional and inverted instruments.

General term for a laboratory apparatus commonly used for performing polymerase chain reaction (PCR). The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps.

A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. The instrument is designed to measure the amount of stimulated electromagnetic radiation produced by pulses of electromagnetic radiation emitted into a water sample or in situ.

General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.

Project Description

Heterotrophy supports much of the energy-starved life in the deep biosphere, yet we know almost nothing about the pathways by which organic matter (OM) is remineralized in the deep subseafloor. The microbes responsible for OM breakdown in the subsurface are taxonomically diverse, but their lack of resemblance to well-characterized lab cultures makes it impossible to use taxonomy alone to predict the nature of their interactions with OM. Even when metagenomes or metatranscriptomes are available to provide functional information, common analysis tools only categorize annotated genes into general classes that do not distinguish between degradative, synthetic, or housekeeping processes. We propose to develop detailed gene homologue analyses to unlock hidden details about OM degradation pathways in subsurface metagenomes from a range of OM types and amounts in the Peru Margin deep biosphere. This will allow us to describe the intricate landscape of biological remineralization of OM in the marine subsurface. Finally, we will develop an open-source software tool that replicates our analysis methods (freely available to the community via web-interface or as source code from a public repository such as GitHub) to allow other researchers to perform this analysis automatically.

Acquisition Description

The cores were sequentially cut into 3 cm section from the topmost to bottommost depth. For methane measurements, 3 ml of sediments were taken via cut-off syringe immediately after each section was sliced and quickly added to 60 ml serum vials containing 1 ml of 0.1 M KOH, which were stoppered and crimp-sealed with butyl rubber stoppers to minimize gas loss. After being shaken for 1 min to release methane from sediments (> 99.5% of the methane equilibrated in the headspace), a 5 ml headspace aliquot was displaced with an equal volume of anaerobic distilled water, injected into a 1 ml sample loop, and then analyzed on an Agilent 7890a gas chromatograph equipped with flame ionization detector. For sulfate measurements, plastic 15 ml tubes filled completely with sediment were centrifuged and the resulting porewater was filtered at 0.2 µm, acidified with 10% HCl and measured using a 2010i Dionex ion chromatograph.

Processing Description

Methane concentrations (mmol per litre of porewater) were calculated using the following equation:
[CH4] = (ρ(CH4)Vheadspace)/(RTφVsed1000)
where p(CH4) is the partial pressure of methane (in ppmv), Vheadspace is the volume of the serum vial headspace (ml) after the sediment and KOH are added, R is the universal gas constant, T is the temperature at time of measurement in Kelvin and Vsed is the volume (ml) of whole sediment added to the serum vial.

Porosity, φ, was calculated using the formula:
φ = (mw/ ρw)/(mw/ρw+((md-S*mw/1000)/ρds))
where mw is the mass of the water lost on drying, md is the mass of the dried sediment, ρw is the density of pure water, ρds is the density of dry sediment (assumed to be 2.5 g cm−3), and S is salinity in grams per kilogram (assumed to be 19 grams per kilogram for all samples).

Standards at sulfate concentrations 0, 0.1, 0.5, 1, 5, 10 mM measure prior to samples from each core and sample peak areas were converted to sulfate concentrations using the standard curves after accounting for the dilution ((peak area * slope + intercept) * 0.7 / 0.6) by the 10% HCl.

Instruments

Instrument separating gases, volatile substances, or substances dissolved in a volatile solvent by transporting an inert gas through a column packed with a sorbent to a detector for assay. (from SeaDataNet, BODC)

Ion chromatography is a form of liquid chromatography that measures concentrations of ionic species by separating them based on their interaction with a resin. Ionic species separate differently depending on species type and size. Ion chromatographs are able to measure concentrations of major anions, such as fluoride, chloride, nitrate, nitrite, and sulfate, as well as major cations such as lithium, sodium, ammonium, potassium, calcium, and magnesium in the parts-per-billion (ppb) range. (from http://serc.carleton.edu/microbelife/research_methods/biogeochemical/ic.html)

When used in a JGOFS/GLOBEC dataset the depth is a best estimate; usually but not always calculated from pressure; calculated either from CTD pressure using Fofonoff and Millard (1982; UNESCO Tech Paper #44) algorithm adjusted for 1980 equation of state for seawater (EOS80) or simply equivalent to nominal depth as recorded during sampling if CTD pressure was unavailable.

Project Description

Marine sediments contain a microbial population large enough to rival that of Earth's oceans, but much about this vast community is unknown. Innovations in total cell counting methods have refined estimates of cell concentrations, but tell us nothing about specific taxa. Isotopic data provides evidence that a majority of subsurface microorganisms survive by breaking down organic matter, yet measurable links between specific microbial taxa and their organic matter substrates are untested. The proposed work overcomes these limitations, with a particular focus on the degradation of proteins and carbohydrates, which comprise the bulk of classifiable sedimentary organic matter. The project will link specific taxa to potential extracellular enzyme activity in the genomes of single microbial cells, apply newly-identified, optimal methods for counting viable cells belonging to specific taxa using catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH), and measure the potential activity of their enzymes in situ. The resulting data will provide key evidence about the strategies subsurface life uses to overcome extreme energy limitation and contribute to the long-term carbon cycle.

The Principal Investigators are employing novel,improved methods to quantify cells of specific taxa in the marine subsurface and to determine the biogeochemical functions of those uncultured taxa, including:
1) Determine the pathway of organic carbon degradation in single cell genomes of uncultured, numerically dominant subsurface microorganisms.
2) Quantify viable bacteria and archaea in the deep subsurface using an improvement on the existing technology of CARD-FISH.
3 )Measure the potential activities (Vmax values) of enzymes in deep Baltic Sea sediments, and use the abundances of enzyme-producing microorganisms to calculate depth profiles of cell-specific Vmax values.

The project combines these methods in order to identify and quantify the cells capable of degrading organic matter in deep sediments of the Baltic Sea, obtained from Integrated Ocean Drilling Program (IODP) expedition 347. These results will greatly expand our knowledge of the function and activity of uncultured microorganisms in the deep subsurface.

When used in a JGOFS/GLOBEC dataset the depth is a best estimate; usually but not always calculated from pressure; calculated either from CTD pressure using Fofonoff and Millard (1982; UNESCO Tech Paper #44) algorithm adjusted for 1980 equation of state for seawater (EOS80) or simply equivalent to nominal depth as recorded during sampling if CTD pressure was unavailable.

Project Description

Marine sediments contain a microbial population large enough to rival that of Earth's oceans, but much about this vast community is unknown. Innovations in total cell counting methods have refined estimates of cell concentrations, but tell us nothing about specific taxa. Isotopic data provides evidence that a majority of subsurface microorganisms survive by breaking down organic matter, yet measurable links between specific microbial taxa and their organic matter substrates are untested. The proposed work overcomes these limitations, with a particular focus on the degradation of proteins and carbohydrates, which comprise the bulk of classifiable sedimentary organic matter. The project will link specific taxa to potential extracellular enzyme activity in the genomes of single microbial cells, apply newly-identified, optimal methods for counting viable cells belonging to specific taxa using catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH), and measure the potential activity of their enzymes in situ. The resulting data will provide key evidence about the strategies subsurface life uses to overcome extreme energy limitation and contribute to the long-term carbon cycle.

The Principal Investigators are employing novel,improved methods to quantify cells of specific taxa in the marine subsurface and to determine the biogeochemical functions of those uncultured taxa, including:
1) Determine the pathway of organic carbon degradation in single cell genomes of uncultured, numerically dominant subsurface microorganisms.
2) Quantify viable bacteria and archaea in the deep subsurface using an improvement on the existing technology of CARD-FISH.
3 )Measure the potential activities (Vmax values) of enzymes in deep Baltic Sea sediments, and use the abundances of enzyme-producing microorganisms to calculate depth profiles of cell-specific Vmax values.

The project combines these methods in order to identify and quantify the cells capable of degrading organic matter in deep sediments of the Baltic Sea, obtained from Integrated Ocean Drilling Program (IODP) expedition 347. These results will greatly expand our knowledge of the function and activity of uncultured microorganisms in the deep subsurface.

Acquisition Description

Sampling and Analytical Methodology:
Genomic DNA was extracted from Baltic Sea Basin sediments using FastDNA® Spin Kit for Soil
(MP Biomedicals). 16S rRNA gene copy numbers of targets were quantified with qPCR using the
primers in the table in datasheet. Results of qPCR were rejected if the R2 of the standard curve was
below 0.95, or if the melt curve showed evidence of primer dimers. SYBR green chemistry was used
for all reactions, and Invitrogen mastermix was used for DNA copy number measurement on a
BioRad iQ5 (Applied Biosystems, Foster City, California). Serial dilutions of full-length 16S rRNA
gene PCR products from plasmids containing amplified partial 16S genes were used as standards.

Processing Description

Data Processing:
Absolute quantification was calculated by converting Ct values of samples into copy numbers per
microliter of DNA with the linear equation produced by the standard curve with R2 greater than 0.95.
The quantification limit was defined as having fluorescence threshold cycle numbers (Ct) well within
those of the simultaneously-run standard curve and being at least 3 Ct below the non-template control
Ct.

Instruments

Genomic DNA was extracted from Baltic Sea Basin sediments using FastDNA® Spin Kit for Soil
(MP Biomedicals). 16S rRNA gene copy numbers of targets were quantified with qPCR using the
primers in the table in datasheet. Results of qPCR were rejected if the R2 of the standard curve was
below 0.95, or if the melt curve showed evidence of primer dimers. SYBR green chemistry was used
for all reactions, and Invitrogen mastermix was used for DNA copy number measurement on a
BioRad iQ5 (Applied Biosystems, Foster City, California). Serial dilutions of full-length 16S rRNA
gene PCR products from plasmids containing amplified partial 16S genes were used as standards.

General term for a laboratory apparatus commonly used for performing polymerase chain reaction (PCR). The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps.

Genomic DNA was extracted from Baltic Sea Basin sediments using FastDNA® Spin Kit for Soil
(MP Biomedicals). 16S rRNA gene copy numbers of targets were quantified with qPCR using the
primers in the table in datasheet. Results of qPCR were rejected if the R2 of the standard curve was
below 0.95, or if the melt curve showed evidence of primer dimers. SYBR green chemistry was used
for all reactions, and Invitrogen mastermix was used for DNA copy number measurement on a
BioRad iQ5 (Applied Biosystems, Foster City, California). Serial dilutions of full-length 16S rRNA
gene PCR products from plasmids containing amplified partial 16S genes were used as standards.

General term for a laboratory apparatus commonly used for performing polymerase chain reaction (PCR). The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps.

Project Description

Marine sediments contain a microbial population large enough to rival that of Earth's oceans, but much about this vast community is unknown. Innovations in total cell counting methods have refined estimates of cell concentrations, but tell us nothing about specific taxa. Isotopic data provides evidence that a majority of subsurface microorganisms survive by breaking down organic matter, yet measurable links between specific microbial taxa and their organic matter substrates are untested. The proposed work overcomes these limitations, with a particular focus on the degradation of proteins and carbohydrates, which comprise the bulk of classifiable sedimentary organic matter. The project will link specific taxa to potential extracellular enzyme activity in the genomes of single microbial cells, apply newly-identified, optimal methods for counting viable cells belonging to specific taxa using catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH), and measure the potential activity of their enzymes in situ. The resulting data will provide key evidence about the strategies subsurface life uses to overcome extreme energy limitation and contribute to the long-term carbon cycle.

The Principal Investigators are employing novel,improved methods to quantify cells of specific taxa in the marine subsurface and to determine the biogeochemical functions of those uncultured taxa, including:
1) Determine the pathway of organic carbon degradation in single cell genomes of uncultured, numerically dominant subsurface microorganisms.
2) Quantify viable bacteria and archaea in the deep subsurface using an improvement on the existing technology of CARD-FISH.
3 )Measure the potential activities (Vmax values) of enzymes in deep Baltic Sea sediments, and use the abundances of enzyme-producing microorganisms to calculate depth profiles of cell-specific Vmax values.

The project combines these methods in order to identify and quantify the cells capable of degrading organic matter in deep sediments of the Baltic Sea, obtained from Integrated Ocean Drilling Program (IODP) expedition 347. These results will greatly expand our knowledge of the function and activity of uncultured microorganisms in the deep subsurface.

Abstract

Two common quantification methods for subseafloor microorganisms are catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and quantitative PCR (qPCR). Using these methods, we quantified Bacteria and Archaea in Baltic Sea basin sediments (IODP Exp. 347) down to 90 mbsf, testing the following hypotheses in an inter-laboratory comparison: 1) proteinase K permeabilization of Archaeal cell walls increases CARD-FISH accuracy, and 2) qPCR varies by more than an order of magnitude between laboratories using similar protocols. CARD-FISH counts did not differ between permeabilization treatments, demonstrating that proteinase K did not increase accuracy of CARD-FISH counts. However, 91% of these counts were below the quantification limit of 1.3 × 107 cells cm−3. For qPCR, data varied between laboratories, but were largely within the same order of magnitude if the same primers were used, with 88% of samples being above the quantification limit. Copy number values were elevated by preparing a sediment slurry before DNA extraction: 3.88 ×106 to 2.34 ×109 16S rRNA gene copies cm−3 vs. 1.39 × 107 to 1.87 × 109 total cells cm−3. By qPCR, Bacteria were more abundant than Archaea, although they usually were within the same order of magnitude. Overall, qPCR is more sensitive than CARD-FISH, but both require optimization to consistently achieve both precision and accuracy.

Abstract

A genomic reconstruction belonging to the genus Methanosarcina was assembled from metagenomic data from a methane-producing enrichment of Antarctic permafrost. This is the first methanogen genome reported from permafrost of the Dry Valleys and can help shed light on future climate-affected methane dynamics.

Abstract

The hydrothermal sediments of Guaymas Basin, an active spreading center in the Gulf of California (Mexico), are rich in porewater methane, short-chain alkanes, sulfate and sulfide, and provide a model system to explore habitat preferences of microorganisms, including sulfate-dependent, methane- and short chain alkane-oxidizing microbial communities. In this study, hot sediments (above 60°C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed “Mat Mound”) were characterized by porewater geochemistry of methane, C2–C6 short-chain alkanes, sulfate, sulfide, sulfate reduction rate measurements, in situ temperature gradients, bacterial and archaeal 16S rRNA gene clone libraries and V6 tag pyrosequencing. The most abundantly detected groups in the Mat mound sediments include anaerobic methane-oxidizing archaea of the ANME-1 lineage and its sister clade ANME-1Guaymas, the uncultured bacterial groups SEEP-SRB2 within the Deltaproteobacteria and the separately branching HotSeep-1 Group; these uncultured bacteria are candidates for sulfate-reducing alkane oxidation and for sulfate-reducing syntrophy with ANME archaea. The archaeal dataset indicates distinct habitat preferences for ANME-1, ANME-1-Guaymas, and ANME-2 archaea in Guaymas Basin hydrothermal sediments. The bacterial groups SEEP-SRB2 and HotSeep-1 co-occur with ANME-1 and ANME-1Guaymas in hydrothermally active sediments underneath microbial mats in Guaymas Basin. We propose the working hypothesis that this mixed bacterial and archaeal community catalyzes the oxidation of both methane and short-chain alkanes, and constitutes a microbial community signature that is characteristic for hydrothermal and/or cold seep sediments containing both substrates.

Project Description

Marine sediments contain a microbial population large enough to rival that of Earth's oceans, but much about this vast community is unknown. Innovations in total cell counting methods have refined estimates of cell concentrations, but tell us nothing about specific taxa. Isotopic data provides evidence that a majority of subsurface microorganisms survive by breaking down organic matter, yet measurable links between specific microbial taxa and their organic matter substrates are untested. The proposed work overcomes these limitations, with a particular focus on the degradation of proteins and carbohydrates, which comprise the bulk of classifiable sedimentary organic matter. The project will link specific taxa to potential extracellular enzyme activity in the genomes of single microbial cells, apply newly-identified, optimal methods for counting viable cells belonging to specific taxa using catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH), and measure the potential activity of their enzymes in situ. The resulting data will provide key evidence about the strategies subsurface life uses to overcome extreme energy limitation and contribute to the long-term carbon cycle.

The Principal Investigators are employing novel,improved methods to quantify cells of specific taxa in the marine subsurface and to determine the biogeochemical functions of those uncultured taxa, including:
1) Determine the pathway of organic carbon degradation in single cell genomes of uncultured, numerically dominant subsurface microorganisms.
2) Quantify viable bacteria and archaea in the deep subsurface using an improvement on the existing technology of CARD-FISH.
3 )Measure the potential activities (Vmax values) of enzymes in deep Baltic Sea sediments, and use the abundances of enzyme-producing microorganisms to calculate depth profiles of cell-specific Vmax values.

The project combines these methods in order to identify and quantify the cells capable of degrading organic matter in deep sediments of the Baltic Sea, obtained from Integrated Ocean Drilling Program (IODP) expedition 347. These results will greatly expand our knowledge of the function and activity of uncultured microorganisms in the deep subsurface.

Project Description

Heterotrophy supports much of the energy-starved life in the deep biosphere, yet we know almost nothing about the pathways by which organic matter (OM) is remineralized in the deep subseafloor. The microbes responsible for OM breakdown in the subsurface are taxonomically diverse, but their lack of resemblance to well-characterized lab cultures makes it impossible to use taxonomy alone to predict the nature of their interactions with OM. Even when metagenomes or metatranscriptomes are available to provide functional information, common analysis tools only categorize annotated genes into general classes that do not distinguish between degradative, synthetic, or housekeeping processes. We propose to develop detailed gene homologue analyses to unlock hidden details about OM degradation pathways in subsurface metagenomes from a range of OM types and amounts in the Peru Margin deep biosphere. This will allow us to describe the intricate landscape of biological remineralization of OM in the marine subsurface. Finally, we will develop an open-source software tool that replicates our analysis methods (freely available to the community via web-interface or as source code from a public repository such as GitHub) to allow other researchers to perform this analysis automatically.

Abstract

The recently proposed candidatus order Altiarchaeales remains an uncultured archaeal lineage composed of genetically diverse, globally widespread organisms frequently observed in anoxic subsurface environments. In spite of 15 years of studies on the psychrophilic biofilm-producingCandidatus Altiarchaeum hamiconexum and its close relatives, very little is known about the phylogenetic and functional diversity of the widespread free-living marine members of this taxon. From methanogenic sediments in the White Oak River Estuary, NC, USA, we sequenced a single cell amplified genome (SAG), WOR_SM1_SCG, and used it to identify and refine two high-quality genomes from metagenomes, WOR_SM1_79 and WOR_SM1_86-2, from the same site. These three genomic reconstructions form a monophyletic group, which also includes three previously published genomes from metagenomes from terrestrial springs and a SAG from Sakinaw Lake in a group previously designated as pMC2A384. A synapomorphic mutation in the Altiarchaeales tRNA synthetase β subunit, pheT, caused the protein to be encoded as two subunits at non-adjacent loci. Consistent with the terrestrial spring clades, our estuarine genomes contained a near-complete autotrophic metabolism, H2 or CO as potential electron donors, a reductive acetyl-CoA pathway for carbon fixation, and methylotroph-like NADP(H)-dependent dehydrogenase. Phylogenies based on 16S rRNA genes and concatenated conserved proteins identified two distinct sub-clades of Altiarchaeales, Alti-1 populated by organisms from actively flowing springs, and Alti-2 which was more widespread, diverse, and not associated with visible mats. The core Alti-1 genome suggested Alti-1 is adapted for the stream environment with lipopolysaccharide production capacity and extracellular hami structures. The core Alti-2 genome suggested members of this clade are free-living with distinct mechanisms for energy maintenance, motility, osmoregulation, and sulfur redox reactions. These data suggested that the hamus structures found in Candidatus Altiarchaeum hamiconexum are not present outside of stream-adapted Altiarchaeales. Homologs to a Na+transporter and membrane bound coenzyme A disulfide reductase that were unique to the brackish sediment Alti-2 genomes, could indicate adaptations to the estuarine, sulfur-rich environment.

Abstract

The hydrothermal mats, mounds, and chimneys of the southern Guaymas Basin are the surface expression of complex subsurface hydrothermal circulation patterns. In this overview, we document the most frequently visited features of this hydrothermal area with photographs, temperature measurements, and selected geochemical data; many of these distinct habitats await characterization of their microbial communities and activities. Microprofiler deployments on microbial mats and hydrothermal sediments show their steep geochemical and thermal gradients at millimeter-scale vertical resolution. Mapping these hydrothermal features and sampling locations within the southern Guaymas Basin suggest linkages to underlying shallow sills and heat flow gradients. Recognizing the inherent spatial limitations of much current Guaymas Basin sampling calls for comprehensive surveys of the wider spreading region.

Abstract

Marine sediments host a large population of diverse, heterotrophic, uncultured microorganisms with unknown physiologies that control carbon flow through organic matter decomposition. Recently, single-cell genomics uncovered new key players in these processes, such as the miscellaneous crenarchaeotal group. These widespread archaea encode putative intra- and extracellular proteases for the degradation of detrital proteins present in sediments. Here, we show that one of these enzymes is a self-compartmentalizing tetrameric aminopeptidase with a preference for cysteine and hydrophobic residues at the N terminus of the hydrolyzed peptide. The ability to perform detailed characterizations of enzymes from native subsurface microorganisms, without requiring that those organisms first be grown in pure culture, holds great promise for understanding key carbon transformations in the environment as well as identifying new enzymes for biomedical and biotechnological applications.

Abstract

The ability of microorganisms to withstand long periods with extremely low energy input has gained increasing scientific attention in recent years. Starvation experiments in the laboratory have shown that a phylogenetically wide range of microorganisms evolve fitness-enhancing genetic traits within weeks of incubation under low-energy stress. Studies on natural environments that are cut off from new energy supplies over geologic time scales, such as deeply buried sediments, suggest that similar adaptations might mediate survival under energy limitation in the environment. Yet, the extent to which laboratory-based evidence of starvation survival in pure or mixed cultures can be extrapolated to sustained microbial ecosystems in nature remains unclear. In this review, we discuss past investigations on microbial energy requirements and adaptations to energy limitation, identify gaps in our current knowledge, and outline possible future foci of research on life under extreme energy limitation.

Abstract

During the past decade, the IODP (International Ocean Discovery Program) has fostered a significant increase in deep biosphere investigations in the marine sedimentary and crustal environments, and scientists are well-poised to continue this momentum into the next phase of the IODP. The goals of this workshop were to evaluate recent findings in a global context, synthesize available biogeochemical data to foster thermodynamic and metabolic activity modeling and measurements, identify regional targets for future targeted sampling and dedicated expeditions, foster collaborations, and highlight the accomplishments of deep biosphere research within IODP. Twenty-four scientists from around the world participated in this one-day workshop sponsored by IODP-MI and held in Florence, Italy, immediately prior to the Goldschmidt 2013 conference. A major topic of discussion at the workshop was the continued need for standard biological sampling and measurements across IODP platforms. Workshop participants renew the call to IODP operators to implement recommended protocols.

Abstract

Half of the microbial cells in the Earth’s oceans are found in sediments. Many of these cells are members of the Archaea, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota and Aigarchaeota, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.

Abstract

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.

One of the major challenges in the study of deep subsurface microbiology is determining the geochemical processes performed by uncultured microorganisms. These microorganisms are often abundant and diverse, but have been identified only by taxonomic marker genes. We addressed this issue by sequencing whole genomes from individual cells of key groups of uncultured archaea and bacteria. This allowed us to link metabolic functions and phylogenetic identities to each other as well as to key environmental factors such as the source of organic matter. By accessing samples from IODP Leg 347: Baltic Sea Paleoenvironment, we compared whole genomes from individual cells across glacial and interglacial periods. We found that the most abundant bacteria and archaea in sediments up to 85 meters below the seafloor were able to be sorted with flow cytometry and sequenced, yielding new single cell genomes from deeply-branching clades. Different clades had different sets of genes, that were conserved between single cell genomes of the same clade. Quantitative PCR showed that bacteria were dominant over archaea in these sediments. We continue to explore the links between microbial phylogeny and function in these deep subsurface prokaryotic communities.

Current Placement: Assistant Professor, University of Tennessee, 2014-

Amount: $148,422.00

Award Dates: September 1, 2013 — October 31, 2015

Abstract

Heterotrophy supports much of the energy-starved life in the deep biosphere, yet we know almost nothing about the pathways by which organic matter (OM) is remineralized in the deep subseafloor. The microbes responsible for OM breakdown in the subsurface are taxonomically diverse, but their lack of resemblance to well-characterized lab cultures makes it impossible to use taxonomy alone to predict the nature of their interactions with OM. Even when metagenomes or metatranscriptomes are available to provide functional information, common analysis tools only categorize annotated genes into general classes that do not distinguish between degradative, synthetic, or housekeeping processes. We approached this problem by expressing, purifying, characterizing, and analyzing proteins from the uncultured phylum, Bathyarchaeota. We explored the possibilities for long term starvation in marine organisms in a review paper, and developed software for data processing of OM degradation. We monitored marine sediments in lab to look for growth dynamics in natural populations of microbes, and quantified the abundance and evolutionary distance of uncultured microbes in the public database. In total, we were able to identify new potential roles for uncultured archaea in OM degradation.